Monoclonal antibody for recognizing HPV16 positive tumor cells, and applications thereof

An antibody and carrier technology, applied in applications, antibodies, anti-tumor drugs, etc., can solve problems such as low sensitivity, inability to obtain immune response, and lack of specificity of E7 protein

Active Publication Date: 2017-02-01
ATTOGEN BIOMEDICAL SUZHOU INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the sensitivity of cytological methods to detect CIN2 and higher-grade lesions is low. Cytology does not have very good specificity for precancerous lesions. - 3 years will be tested
In 2015, the latest guidelines issued by the American Cancer Society (ACS) recommended the use of human papillomavirus (HPV) detection for primary screening, but for areas lacking in health resources in China, this technology has no practical application conditions, and HPV DNA detection cannot distinguish Is HPV transient or persistent?
There are three main reasons why there are no suitable antibodies for clinical HPV detection: 1. The expression of HPV protein in clinical tissue or cell samples is low, and high-affinity antibodies are required for detection; 2. The HPV virus cannot be detected under the existing standard tissue culture techniques. Survival in laboratory culture; 3. The E7 protein itself has immunosuppression, so that the animals immunized with E7 protein cannot obtain a good immune response. In addition, the prepared antibodies often cross-react with other HPV proteins and have no specificity for E7 protein. specificity

Method used

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  • Monoclonal antibody for recognizing HPV16 positive tumor cells, and applications thereof
  • Monoclonal antibody for recognizing HPV16 positive tumor cells, and applications thereof
  • Monoclonal antibody for recognizing HPV16 positive tumor cells, and applications thereof

Examples

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preparation example Construction

[0175] Preparation of monoclonal antibodies

[0176] Antibodies of the present invention can be prepared by various techniques known to those skilled in the art. For example, an antigen of the invention may be administered to an animal to induce the production of monoclonal antibodies. For monoclonal antibodies, hybridoma technology can be used to prepare (see Kohler et al., Nature 256; 495, 1975; Kohler et al., Eur.J.Immunol.6:511, 1976; Kohler et al., Eur.J.Immunol. 6:292,1976; Hammerling et al., In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, N.Y., 1981), phage display technology or available recombinant DNA method (US Patent No. 4,816,567).

[0177]Representative myeloma cells are those that fuse efficiently, support stable high-level production of antibody by selected antibody-producing cells, and are sensitive to culture medium (HAT medium matrix), including myeloma cell lines, such as murine Myeloma cell lines, including those derived from MOPC-21 and MPC-11...

Embodiment 1

[0212] 1. Preparation of rabbit monoclonal antibody against human papillomavirus HPV16 E7

[0213] 1.1 Screening of single-chain antibody (scFv)

[0214] Rabbits were immunized with His-HPV16 E7 recombinant protein, and the titer was detected with His-HPV16 E7 recombinant protein and His irrelevant protein. Isolation of rabbit B lymphocytes to obtain immunoglobulin genes. The complete set of variable region genes of B cells is cloned and assembled into a phage antibody library. The constructed phage antibody library was panned with the recombinant protein His-HPV16 E7. Enrichment after three rounds of panning; determination of phage titer; amplification of phage plaques; DNA sequencing; The recombinant protein His-HPV16 E7 was selected for ELISA detection and screening, and the negative control (N) was set with His irrelevant protein, the Anti-6×His antibody was set as the positive control (P) coated with His antigen, and the blank control (coated with the antigen directly)...

Embodiment 2

[0235] Example 2 Detection of overexpression of biomarkers in tumor cells treated with liquid-based cell fixative using immunocytochemical staining

[0236] Collect CaSki cells and C-33A cells in the logarithmic growth phase by centrifugation, mix Caski cells and C-33A cells at a ratio of 1:1, and plant them on poly-L-Lysine-treated coverslips at 37°C, 5% CO 2 Cultivate for 24h. Carefully rinse twice with PBS after discarding the medium. The coverslip was taken out, and liquid-based cell fixative (Hologic, Preserv Solution) fixed solution for several days. Before use, place the coverslip in 99% ethanol for 10 min to 1 h, and air-dry overnight.

[0237] After the air-dried tumor cell coverslips were placed in 50% ethanol for 10 min, they were transferred to deionized water for at least 30 s. To prevent non-specific background staining, do not allow coverslips to dry out during staining. Place the deionized water-treated cell slides in Tris-EDTA (pH 9.0) repair solution, ...

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Abstract

The invention provides a monoclonal antibody for recognizing HPV16 positive cervical epithelial cancer cells, and applications thereof, wherein the antibody can specifically detect the cervical cancer biomarker HPV16E7 protein in tumor cells so as to distinguish the cancerous cervical epithelial cells and the cervical abnormality or non-cancerous cervical epithelial cells, such that the basis can be provided for doctors so as to accurately diagnose the cancer caused by HPV infection, the missed diagnosis rate of the high-grade cervical lesion can be effectively reduced, the sufficient time and the basis can be provided for the clinical doctor to diagnose and treat the patients, and the early cervical disease detection and the early intervention can be improved; and with the monoclonal antibody, the unnecessary colposcopy can be reduced and avoided.

Description

technical field [0001] The invention belongs to the field of biological diagnosis and medicine. Specifically, the invention relates to a monoclonal antibody for identifying human papillomavirus (HPV) subtype 16 positive tumor cells, including human cervical epithelial cancer cells, and its application. Background technique [0002] Cervical cancer is the second most common female malignancy. About 500,000 women are diagnosed with cervical cancer every year in the world, and more than half of them die from it. After the German scientist Harald Zur Hausen proposed that HPV might be a sexually transmitted carcinogen in 1976, the research on the relationship between HPV infection and cervical cancer became a hot topic in the etiology of tumor viruses. In 2008, Harald zur Hausen won the Nobel Prize in Physiology or Medicine for demonstrating that the HPV virus is the causative agent of cervical cancer. A large number of studies have found that HPV is not only the culprit of cerv...

Claims

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Application Information

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IPC IPC(8): C07K16/08C12N15/13C12N15/63A61K39/395A61P31/20A61P35/00G01N33/571G01N33/574
Inventor 常小迦韩凤丽时成龙
Owner ATTOGEN BIOMEDICAL SUZHOU INC
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